Phosphoserine phosphatase of coryneform bacteria and variants thereof

ABSTRACT

The present invention provides a DNA coding for a protein defined in the following (A) or (B) is obtained from  Brevibacterium flavum  chromosomal DNA library by cloning a DNA fragment that complicates serB deficiency of  Escherichia coli  as a open reading frame in the DNA fragment.
         (A) A protein which comprises an amino acid sequence of SEQ ID: 2 in Sequence Listing; or   (B) A protein which comprises an amino acid sequence including substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 in Sequence Listing, and which has phosphoserine phosphatase activity.

This application is a divisional of U.S. application Ser. No.10/321,382, filed on Dec. 18, 2002 (now U.S. Pat. No. 7,029,896), whichis a continuation of U.S. application Ser. No. 10/081,859, filed on Feb.25, 2002 now abandoned, which is a continuation of U.S. application Ser.No. 09/761,716, filed on Jan. 18, 2001 (now U.S. Pat. No. 6,395,528),which claims priority to JP 2000-23341, filed on Jan. 27, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the DNA coding for phosphoserinephosphatase of coryneform bacteria. The DNA may be utilized formicrobiologic industry, such as breeding L-serine producing coryneformbacteria.

2. Description of the Related Art

As a conventional method of producing L-serine by fermentation, therehas been reported the method in which a bacterial strain capable ofconverting glycine and sugar into L-serine is used in a mediumcontaining 30 g/L of glycine to produce at most 14 g/L of L-serine. Theconversion yield amounted to 46% (Kubota, K., Agricultural BiologicalChemistry, 49, 7-12 (1985)). Using a bacterial strain capable ofconverting glycine and methanol into L-serine, 53 g/L of L-serine can beproduced from 100 g/L of glycine (T. Yoshida et al., Journal ofFermentation and Bioengineering, Vol. 79, No. 2, 181-183, 1995). In themethod using Nocardia, it has been known that the L-serine productivityof the bacterium can be improved by breeding those strains resistant toserine hydroxamate, azaserine or the like (Japanese Patent PublicationNo. 57-1235). However, these methods involve use of glycine that is aprecursor of L-serine and include complicated operation and isdisadvantageous from the viewpoint of costs.

As strains that can ferment L-serine directly from a sugar and do notneed addition of the precursor of L-serine to the medium, there has beenknown Corynebacterium glutamicum that is resistant to D-serine,a-methylserine, o-methylserine, isoserine, serine hydroxamate, and3-chloroalanine but the accumulation of L-serine is as low as 0.8 g/L(Nogei Kagakukaishi, Vol. 48, No. 3, p. 201-208, 1974). Accordingly,further strain improvements are needed for direct fermentation ofL-serine on an industrial scale.

On the other hand, regarding coryneform bacteria, there have beendisclosed a vector plasmid that is capable of autonomous replication inthe cell and having a drug resistance marker gene (cf. U.S. Pat. No.4,514,502) and a method of introducing a gene into the cell (JapanesePatent Application Laid-open No. 2-207791). These techniques have beenutilized for breeding L-amino acid producing bacteria. As for L-serine,it has been found that L-serine productivity of coryneform bacteriahaving the L-serine producing ability is improved by introduction of agene coding for D-3-phosphoglyceratedehydrogenase whose feedbackinhibition by L-serine is desensitized (serA gene) (European PatentApplication Laid-Open No. 943687), or amplification of a gene coding forphosphoserine phosphatase (serB) or phosphoserine transaminase (serC)(European Patent Application Laid-Open No. 931833). There has been knownserB gene in Escherichia coli (GenBank accession X03046, M30784), Yeast(GenBank accession U36473), Helicobacter pylori (GenBank accessionAF006039), however, serB gene of coryneform bacteria has not been known.

SUMMARY OF THE INVENTION

An object of the present invention, in view of the aforementionedpoints, is to provide the DNA coding for phosphoserine phosphatase ofcoryneform bacteria.

The present inventors obtained the DNA fragment from a chromosome DNAlibrary of Brevibacterium flavum, which complimented serB deficiency ofEscherichia coli. The open reading frame having homology with known serBgene of Escherichia coli was subcloned from the DNA fragment andintroduced into serB deficient mutant strain of aforementionedEscherichia coli. However, serB deficiency was not complemented. It wasfound that serB deficiency was complemented with the aforementioned ORFwhich was forcedly expressed utilizing lac promoter. Thus, it wasconfirmed that the aforementioned ORF was serB homologue ofBrevibacterium flavum. It was indicated that the aforementioned ORF didnot have its own promoter because of forming operon.

The present invention was accomplished as described above, and providesthe followings.

(1) A protein defined in the following (A) or (B):

(A) A protein which comprises an amino acid sequence of SEQ ID: 2 inSequence Listing; or

(B) A protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition or inversion of one orseveral amino acids in the amino acid sequence of SEQ ID NO: 2 inSequence Listing, and which has phosphoserine phosphatase activity.

(2) A DNA coding for a protein as defined in the following (A) or (B):

(A) A protein which comprises an amino acid sequence of SEQ ID: 2 inSequence Listing; or

(B) A protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition or inversion of one orseveral amino acids in the amino acid sequence of SEQ ID NO: 2 inSequence Listing, and which has phosphoserine phosphatase activity.

(3) A DNA coding for a protein having phosphoserine phosphataseactivity, which hybridizes under stringent conditions to a DNA sequenceencoding a protein which comprises an amino acid sequence of SEQ ID NO:2.

(4) The DNA according to (3), the stringent conditions comprise washingat 60° C. and at a salt concentration corresponding to 1×SSC and 0.1%SDS.

(5) The DNA according to (2), which is DNA as defined in the following(a) or (b):

(a) A DNA which comprises a nucleotide sequence corresponding tonucleotide numbers of 210-1547 of nucleotide sequence of SEQ NO: 1 inSequence Listing; or

(b) A DNA which is hybridizable with a probe which comprises thenucleotide sequence corresponding to nucleotide numbers of 210-1547 ofnucleotide sequence of SEQ NO: 13 in Sequence Listing or a partialnucleotide sequence under stringent conditions, and which codes for theprotein having the phosphoserine phosphatase activity.

(6) The DNA according to (5), the stringent conditions comprise washingat 60° C. and at a salt concentration corresponding to 1×SSC and 0.1%SDS.

(7) A vector comprising the DNA according to any of (1) to (6).

(8) A bacterial cell in which phosphoserine phosphatase activity encodedby the DNA according to any of (1) to (16) is increased.

(9) A method of producing L-serine comprising the steps of cultivatingthe bacterium according to (8) in a medium to produce and accumulateL-serine in the medium, and collecting L-serine from the medium.

Hereafter, the present invention will be explained in detail.

The DNA of the present invention can be obtained through PCR (polymerasechain reaction) utilizing chromosomal DNA of Brevibacterium flavum, forexample, the Brevibacterium flavum strain ATCC14067, as a template, aswell as a primer having the nucleotide sequence of SEQ ID NOs: 3 and 4shown in sequence listing. Because each of these primers has arestriction enzyme recognition site of EcoRI or SalI in their 5′sequences, the amplification product digested with these restrictionenzymes can be inserted into a vector having EcoRI and SalI digestedends.

The nucleotide sequences of the aforementioned primers were designedbased on the nucleotide sequence of the DNA fragment which complementsserB deficient mutant strain Escherichia coli ME8320 (thi, serB,zhi-1::Tn10) (available from national genetics institute). By usingthese primers, a DNA fragment containing the coding region of the serBhomologue and its flanking region (5′ non-translation region of about200 bp and 3′ non-translation region of about 300 bp) can be obtained.

The nucleotide sequence of the coding region of the DNA of the presentinvention obtained as described above and an amino acid sequence whichmay be encoded by the sequence are shown in SEQ ID NO: 1. The amino acidsequence alone is shown in SEQ ID NO: 2.

The aforementioned serB homologue was found as the open reading frame(ORF) having homology with serB genes of Escherichia coli and Yeast(Saccharomyces cerevisiae), which existed in the DNA fragmentcomplementing serB deficiency of strain ME8320. The DNA fragment havingenough length to contain the ORF and the promoter was obtained fromBrevibacterium flavum ATCC14067 by PCR utilizing aforementioned primershaving the nucleotide sequence of SEQ ID NOs: 3 and 4 shown in sequencelisting. It was introduced into strain ME8320, however the serBdeficiency of the strain was not complemented. Therefore, at first, itwas thought that the aforementioned ORF was not serB homologue. However,the serB deficiency was complemented with the aforementioned ORF whichwas ligated to lac promoter and forcedly expressed. Thus, it wasconfirmed that the aforementioned ORF was serB homologue.

Further, the other ORF was found just upstream of serB homologue in theDNA fragment that compliments serB deficiency. From these results, itwas indicated that these ORFs form a operon and there was no promoterregion and the like just upstream of serB homologue.

At first, it was attempted to obtain serB homologue of Brevibacteriumflavum utilizing nucleotide sequence of known serB gene. That is, theinventors intended to compare nucleotide sequence and amino acidsequence of known serB gene among the other species, to search highlyconserved amino acid sequence region among various species, tosynthesize PCR primers based on the nucleotide sequence of the regionand to amplify serB homologue with these primers. However, since therewere few such conserved regions, they estimated that it was difficult toobtain the objective gene by PCR. Therefore, complementation testutilizing serB deficient mutant strain was performed.

While the DNA of the present invention was obtained by cloning bycomplementation test utilizing serB deficient mutant and subcloning byPCR as described above, it can also be obtained from a chromosome DNAlibrary of Brevibacterium flavum by hybridization utilizing anoligonucleotide as a probe prepared based on the nucleotide sequence ofthe DNA of the present invention.

Methods for construction of genomic DNA library, hybridization, PCR,preparation of plasmid DNA, digestion and ligation of DNA,transformation and the like are described in Sambrook, J., Fritsch, andE. F., Maniatis, T., Molecular Cloning, Cold Spring Harbor LaboratoryPress, 1.21 (1989).

The DNA of the present invention may encode phosphoserine phosphataseincluding substitution, deletion, insertion, addition, or inversion ofone or several amino acids at one or a plurality of positions, providedthat the activity of phosphoserine phosphatase encoded thereby is notdeteriorated. Although the number of “several” amino acids differsdepending on the position or the type of amino acid residues in thethree-dimensional structure of the protein, it may be 2 to 200,preferably 2 to 50, and more preferably 2 to 20.

Further, the DNA of the present invention may encode phosphoserinephosphatase having homology of not less than 50%, preferably not lessthan 60%, more preferably not less than 70%, further preferably not lessthan 80%, and most preferably not less than 90% with the amino acidsequence of SEQ ID NO: 2, provided that the activity of phosphoserinephosphatase encoded thereby is not deteriorated.

DNA, which encodes the substantially same protein as phosphoserinephosphatase as described above, is obtained, for example, by modifyingthe nucleotide sequence of phosphoserine phosphatase gene, for example,by means of the site-directed mutagenesis method so that one or moreamino acid residues at a specified site of the gene involvesubstitution, deletion, insertion, addition, or inversion. DNA modifiedas described above may be obtained by the conventionally known mutationtreatment. The mutation treatment includes a method for treating DNAcoding for phosphoserine phosphatase in vitro, for example, withhydroxylamine, and a method for treating a microorganism, for example, abacterium belonging to the genus Escherichia harboring DNA encodingphosphoserine phosphatase with ultraviolet irradiation or a mutatingagent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrousacid usually used for the mutation treatment.

The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes mutation (mutant or variant)which naturally occurs, for example, the difference in strains, speciesor genera of the microorganism.

The DNA, which codes for substantially the same protein as phosphoserinephosphatase, is obtained by expressing DNA having mutation as describedabove in an appropriate cell, and investigating the phosphoserinephosphatase activity of an expressed product. The DNA, which codes forsubstantially the same protein as phosphoserine phosphatase, is alsoobtained by isolating DNA which is hybridizable with a primer having,for example, the nucleotide sequence comprise the nucleotide numbers of210-1547 of the nucleotide sequence of SEQ ID NO:1, under stringentconditions, and which codes for a protein having the phosphoserinephosphatase activity, from DNA coding for phosphoserine phosphatasehaving mutation or from a cell harboring it. The “stringent conditions”referred to herein are conditions under which so-called specific hybridis formed, and non-specific hybrid is not formed. It is difficult toclearly express this condition by using any numerical value. However,for example, the stringent conditions include conditions under whichDNA's having high homology, for example, DNA's having homology of notless than 50% are hybridized with each other, and DNA's having homologylower than the above are not hybridized with each other. Alternatively,the stringent conditions are exemplified by conditions which compriseordinary condition of washing in Southern hybridization, e.g., 60° C.,1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS.

DNA which has homology of not less than 60%, preferably not less than70%, more preferably not less than 80%, and most preferably not lessthan 90% with the nucleotide sequence of SEQ ID NO: 1, and encodes aprotein having the activity of phosphoserine phosphatase is included inthe DNA of the present invention.

As a probe, a partial sequence of the nucleotide sequence of SEQ ID NO:1 can also be used. Such a probe may be prepared by PCR usingoligonucleotides produced based on the nucleotide sequence of SEQ ID NO:1 as primers, and a DNA fragment containing the nucleotide sequence ofSEQ ID NO: 1 as a template. When a DNA fragment in a length of about 300bp is used as the probe, the conditions of washing for the hybridizationconsist of, for example, 50° C., 2×SSC, and 0.1% SDS.

The gene, which is hybridizable under the condition as described above,includes those having a stop codon generated in the gene, and thosehaving no activity due to mutation of active center. However, suchmutant genes can be easily removed by ligating the gene with acommercially available activity expression vector, and measuring thephosphatase activity in accordance with, for example, the method ofLewis, I. Pizer (J. Biol. Chem., 238(12), 3934-3944 (1963)).

It is preferred that the DNA of the present invention is ligated withvector DNA autonomously replicable in cells of Escherichia coli and/orcoryneform bacteria to prepare recombinant DNA, and the recombinant DNAis introduced into cells of Escherichia coli beforehand. Such provisionmakes following operations easy. The vector autonomously replicable incells of Escherichia coli is preferably a plasmid vector which ispreferably autonomously replicable in cells of a host, including, forexample, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, and RSF1010.

Recombinant DNA may be prepared by utilizing transposon (WO 02/02627International Publication Pamphlet, WO 93/18151 InternationalPublication Pamphlet, European Patent Application Laid-open No. 0445385,Japanese Patent Application Laid-open No. 6-46867, Vertes, A. A. et al.,Mol. Microbiol., 11, 739-746 (1994), Bonamy, C., et al., Mol.Microbiol., 14, 571-581 (1994), Vertes, A. A. et al., Mol. Gen. Genet.,245, 397-405 (1994), Jagar, W. et al., FEMS Microbiology Letters, 126,1-6 (1995), Japanese Patent Application Laid-open No. 7-107976, JapanesePatent Application Laid-open No. 7-327680, etc.), phage vectors,recombination of chromosomes (Experiments in Molecular Genetics, ColdSpring Harbor Laboratory Press (1972); Matsuyama, S. and Mizushima, S.,J. Bacteriol., 162, 1196 (1985)) and the like.

When a DNA fragment having an ability to allow a plasmid to beautonomously replicable in coryneform bacteria is inserted into thesevectors, they can be used as a so-called shuttle vector autonomouslyreplicable in both Escherichia coli and coryneform bacteria.

Such a shuttle vector includes the followings. Microorganisms harboringeach of vectors and deposition numbers in international depositionfacilities are shown in parentheses.

-   -   pHC4: Escherichia coli AJ12617 (FERM BP-3532)    -   pAJ655: Escherichia coli AJ11882 (FERM BP-136), Corynebacterium        glutamicum SR8201 (ATCC 39135)    -   pAJ1844: Escherichia coli AJ11883 (FERM BP-137), Corynebacterium        glutamicum SR8202 (ATCC 39136)    -   pAJ611: Escherichia coli AJ11884 (FERM BP-138)    -   pAJ3148: Corynebacterium glutamicum SR8203 (ATCC 39137)    -   pAJ440: Bacillus subtilis AJ11901 (FERM BP-140)

These vectors are obtainable from the deposited microorganisms asfollows. Cells collected at a logarithmic growth phase were lysed byusing lysozyme and SDS, followed by separation from a lysate bycentrifugation at 30,000×g to obtain a supernatant to which polyethyleneglycol is added, followed by fractionation and purification by means ofcesium chloride-ethidium bromide equilibrium density gradientcentrifugation.

Escherichia coli can be transformed by introducing a plasmid inaccordance with, for example, a method of D. A. Morrison (Methods inEnzymology, 68, 326 (1979)) or a method in which recipient cells aretreated with calcium chloride to increase permeability for DNA (Mandel,M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

Introduction of plasmids to coryneform bacteria to cause transformationcan be performed by the electric pulse method (Sugimoto et al., JapanesePatent Application Laid-open No. 2-207791).

The phosphoserine phosphatase can be produced by expressing the DNA ofthe present invention using a suitable host-vector system.

As a host for the expression of the DNA of the present invention,various prokaryotic cells including bacteria belonging to theCorynebacterium such as Brevibacterium flavum, Escherichia coli,Bacillus subtilis, various eukaryotic cells including Saccharomycescerevisiae, animal cells, and plant cells can be mentioned. Among these,prokaryotic cells, in particular, Escherichia coli and Bacillus subtilisare preferred.

Since the DNA of the present invention does not have a promoter, inorder to express the gene it requires that a promoter which functions inthe host cell, such as lac, trp and P_(L) is ligated to the upstream ofthe DNA sequence. By utilizing a vector containing a promoter as thevector, the ligation of the gene to both vector and promoter can beperformed by one step. As such a vector, pMW219 containing lacZ promoter(available from Nippon gene) can be mentioned.

When the DNA is highly expressed, the plasmid containing the DNA of thepresent invention occasionally becomes unstably. In that case, low copyvector is preferable.

The transformation can be attained by, for example, the method in whichrecipient cells are treated with calcium chloride to increasepermeability for DNA as reported for Escherichia coli K-12 strain(Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the methodutilizing introduction of DNA into competent cells produced from cellsat a growth phase as reported for Bacillus subtilis (Duncan, C. H.,Wilson, G. A., and Young, F. E., Gene, 1, 153 (1977)). It is alsopossible to prepare a protoplast or spheroplast of DNA recipient cell,which readily incorporates DNA, and introduce DNA into it as known forBacillus subtilis, Actinomycetes and yeast (Changs, S. and Choen, S. N.,Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. andHopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B., andFink, G. R., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)). Electricpulse method is also effective for coryneform bacteria (Japanese PatentApplication Laid-open No. 2-207791). The method can be suitably selectedfrom these depending on the cell to be used as the host.

The phosphoserine phosphatase can be produced by culturing cells towhich the DNA of the present invention is introduced in such a mannerthat the DNA can be expressed as described above in a medium to produceand accumulate phosphoserine phosphatase in the culture, and collectingthe phosphoserine phosphatase from the culture. The culture medium canbe selected according to the host to be used.

The phosphoserine phosphatase produced as described above can bepurified from a cell extract or medium as required by using a usualpurification method for enzymes, for example, ion exchangechromatography, gel filtration chromatography, adsorptionchromatography, solvent precipitation and the like.

Further, the DNA of present invention may be utilized for breedingL-serine producing bacteria belonging to coryneform bacteria or thelike. That is, by conferring or enhancing phosphoserine phosphataseactivity by introducing the DNA of present invention into bacterium in aform that the DNA can be expressed, L-serine productivity is conferredand enhanced. Moreover, enhancement of phosphoserine phosphataseactivity can be also performed by amplifying copy numbers of serBhomologue and modifying expression control sequence in order to enhanceexpression of serB homologue in chromosome of Brevibacterium flavum.Modification of expression control sequence in chromosomal DNA isperformed by, for example, substituting strong expression controlsequence such as promoter and the like for that of the operon containingserB homologue (Japanese Patent Application Laid-open No. 1-215280).

Examples of the coryneform bacterium may be used for breeding L-aminoacid producing bacteria include, for example, the following wild typestrains:

-   -   Corynebacterium acetoacidophilum ATCC 13870;    -   Corynebacterium acetoglutamicum ATCC 15806;    -   Corynebacterium callunae ATCC 15991;    -   Corynebacterium glutamicum ATCC 13032;    -   (Brevibacterium divaricatum) ATCC 14020;    -   (Brevibacterium lactofermentum) ATCC 13869;    -   (Corynebacterium lilium) ATCC 15990;    -   Brevibacterium flavum ATCC 14067;    -   Corynebacterium melassecola ATCC 17965;    -   Brevibacterium saccharolyticum ATCC 14066;    -   Brevibacterium immariophilum ATCC 14068;    -   Brevibacterium roseum ATCC 13825;    -   Brevibacterium thiogenitalis ATCC 19240;    -   Microbacterium ammoniaphilum ATCC 15354;    -   Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539).

Mutant strain having resistance to azaserine or β-(2-chenyl)-DL-alanine(European Patent Publication No. 943,687) may be also utilized forbreeding L-serine producing bacteria as a starting strain.

Further, the DNA of present invention may be introduced into L-serineproducing bacteria with other genes of enzyme involving L-serinebiosynthesis. Such genes include the gene coding forD-3-phosphoglyceratedehyidrogenase (serA) (European Patent PublicationNo. 943,687), phosphoserinephosphatase (serB) andphosphoserinetransaminase (serC) (European Patent Publication No.931,833). As serA, mutant gene coding forD-3-phosphoglyceratedehydrogenase whose feedback inhibition by L-serineis desensitized (European Patent Publication No. 943,687).

L-serine may be produced directly from sugars by culturing themicroorganisms to which the DNA of the present invention is introducedin such a form that the DNA can be expressed and which have L-serineproducing ability in a medium to accumulate L-serine in the medium andcollecting L-serine from the medium. Further, the microorganisms towhich the DNA of the present invention is introduced may be applied fora method producing L-serine utilizing L-serine precursor such as glycineand the like, so long as phosphoserine phosphatase is involved in themethod.

For L-serine production using the microorganisms to which the DNA of thepresent invention, the following medium may be used. There can be usedconventional liquid media containing carbon sources, nitrogen sources,inorganic salts, and optionally organic trace nutrients such as aminoacids, vitamins, etc., if desired.

As carbon sources, it is possible to use sugars such as glucose,sucrose, fructose, galactose; saccharified starch solutions, sweetpotato molasses, sugar beet molasses and highest molasses which areincluding the sugars described above; organic acids such as acetic acid;alcohols such as ethanol; glycerol and the like.

As nitrogen sources, it is possible to use ammonia gas, aqueous ammonia,ammonium salts, urea, nitrates and the like. Further, organic nitrogensources for supplemental use, for example, oil cakes, soybeanhydrolysate liquids, decomposed casein, other amino acids, corn steepliquor, yeast or yeast extract, peptides such as peptone, and the like,may be used.

As inorganic ions, phosphoric ion, magnesium ion, calcium ion, iron ion,manganese ion and the like may be added optionally.

In case of using the microorganism of the present invention whichrequires nutrients such as amino acids for its growth, the requirednutrients should be supplemented.

The microorganisms are incubated usually under aerobic conditions at pH5 to 8 and temperature ranges of 25 to 40° C. The pH of the culturemedium is controlled at a predetermined value within the above-describedranges depending on the presence or absence of inorganic or organicacids, alkaline substances, urea, calcium carbonate, ammonia gas, andthe like. L-Serine can be collected from the fermentation liquid, forexample, by separating and removing the cells, subjecting to ionexchange resin treatment, concentration cooling crystallization,membrane separation, and other known methods in any suitablecombination. In order to remove impurities, activated carbon adsorptionand recrystallization may be used for purification.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be more concretely explained with thereference to the following example.

<1> Preparation of Brevibacterium flavum Chromosomal DNA Library Using aHigh Copy Vector

Chromosome was prepared from Brevibacterium flavum ATCC 14067 andpartially digested into approximately 4 to 6 kb fragments withrestriction enzyme Sau3AI. The obtained fragment was ligated toBamHI-digested pSAC which is a shuttle vector of Escherichia coli andcoryneform bacteria.

pSAC4 was prepared as follows.

In order to make a vector pHSG399 for Escherichia coli (Takara Shuzo)autonomously replicable in coryneform bacterium cells, a replicationorigin of the previously obtained plasmid pHM1519 autonomouslyreplicable in coryneform bacterium cells (Miwa, K. et al., Agric. Biol.Chem., 48 (1984) 2901-2903) was introduced into the vector (JapanesePatent Laid-open No. 5-7491). Specifically, pHM1519 was digested withrestriction enzymes BamHI and KpnI to obtain a gene fragment containingthe replication origin, and the obtained fragment was blunt-ended byusing Blunting Lit produced by Takara Shuzo, and inserted into the SalIsite of pHSG399 using a SalI linker (produced by Takara Shuzo) to obtainpSAC4.

Aforementioned ligation reactant was dissolved in TE buffer andEscherichia coli DH5α was transformed using the solution byelectroporation. The transformation solution was added with SOC medium(composition: 20 g/L Bactotrypton, 5 g/L Yeast Extract, 0.5 g/L Nacl, 10g/L glucose), incubated at 37° C. for 1 hour, and then added with anequal volume of 2×LB medium (containing 40 mg/L chloramphenicol,composition of LB medium: 1% Trypton, 0.5% Yeast Extract, 0.1% NaCl,0.1% glucose, pH7) and incubated at 37° C. for 2 hours. The culturemedium was added with equal volume of 4×LB medium containing 40%glycerol and stored at −80° C.

The aforementioned culture medium was inoculated to LB medium andplasmids were collected from obtained cells. The plasmid DNA wasprecipitated with ethanol and transformed into serB deficient mutantstrain ME8320 (thi, serB, zhi-1::Tn10) (obtained from national geneticsinstitute) by electroporation method. It was confirmed that ME8320strain could not glow on the M9 medium containing 140 mg/L vitamin B₁,but could glow on the same medium containing 40 mg/L L-serine.

After transformation, cells were washed, plated on the M9 agar mediumcontaining 40 mg/L vitamin B₁ and chloramphenicol and incubated at 37°C. for 3 to 4 days to form colonies. Plasmids were prepared from eachcolony and examined these size by electophoresis. As a result remarkabledeletion was found. It was considered that high expression of serB genein the cell made plasmid unstably, thus the library should be preparedusing a low copy vector again.

<2> Preparation of Brevibacterium flavum Chromosome DNA Library Using aLow Copy Vector and Cloning of serB Gene

The chromosome was prepared from Brevibacterium flavum ATCC 14067 anddigested with Sau3AI. The reaction was controlled to make the center ofdistribution to be in approximately 3 kbp or more. Approximately 200 μgof obtained digest was separated by 10 to 40% sucrose density gradientcentrifugation and collected as 1 ml fractions with AUTOMATIC LIQUIDCHARG-ER (ADVANTEC) and MICROTUBE PUMP (EYELA). Sucrose density gradientcentrifugation was performed with SW28 rotor (Beckman) at 10° C., 260000rpm, for 26 hours. The fraction containing the DNA fragments that thecenter of distribution was in approximately 3 to 4 kbp or more wasprecipitated with ethanol and purified with Microcon-50 (Milipore).

The chromosomal DNA fragments obtained as described above were ligatedto the low copy vector pMW219 (Nippon gene, BamHI digested anddephosphorylated). The ligation reactant was dissolved in TE buffer andtransformed into Escherichia coli DH5α by electroporation. Thetransformation solution was added with SOC medium, incubated at 37° C.for 1 hour and then added with an equal volume of 2×LB medium(containing 25 mg/L kanamycin) and incubated at 37° C. for 2 hours. Theculture medium was added with equal volume of 4×LB medium containing 40%glycerol and stored at −80° C.

The aforementioned culture medium was inoculated to LB medium andcultivated. Plasmids were collected from obtained cells. The plasmid DNAwas precipitated with ethanol and transformed into serB-deficient mutantstrain ME8320 by electroporation method. After transformation, cellswere washed, plated on the M9 agar medium containing 25 mg/L vitamin B₁and kanamycin and incubated at 37° C. for 3 to 4 days to form colonies.Each colony was plated on the same medium and LB medium containing 25mg/L kanamycin. The strains that could glow on the medium were selectedand plasmids were prepared from the selected strains.

In order to determine nucleotide sequence of the inserted fragment ofthe obtained plasmid, sequencing was started from both ends ofmulti-cloning site of the vector with the universal primers. Thesequencing was forwarded by 300 to 400 bp. Finally approximately 5 kbpof both ends of inserted fragment were sequenced. The open reading framewas searched for determined nucleotide sequence and one ORF havinghomology with phosphoserine phosphatase (coded by serB gene) of otherknown species was found. There were several region showing homology inthe ORF, however, homology between the ORF and known serB was 43% inamino acid sequence and 49.4% in nucleotide sequence for Escherichiacoli and 36.6% in amino acid sequence and 50.9% in nucleotide sequencefor Saccharomyces cerevisiae, respectively. Nucleotide and amino acidsequences were analyzed with the Genetyx-Mac computer program (SoftwareDevelopment Co., Tokyo, Japan). The homology analysis was carried outaccording to the method developed by Lipman and Peason (Science, 227,1435-1441, 1985).

The nucleotide sequence of the ORF having homology with other known serBgene and the flanking regions (SEQ ID No.1), and the amino acid sequencewhich may be encoded by the nucleotide sequence (SEQ ID No.2) are shownin the sequence listing.

<3> Cloning of the ORF Having Homology with serB

The chromosomal DNA fragment containing the ORF and approximately 200 to300 bp of upstream and downstream regions of the ORF was cloned andcomplementation test of serB deficient strain was performed to confirmthat the ORF showing the homology with serB gene was certainly serBgene.

The primers having the nucleotide sequence of SEQ ID Nos: 3 and 4 weredesigned to obtain the desired DNA by PCR. PCR was performed using theseprimers and chromosomal DNA prepared from Brevibacterium flavum ATCC14067 as a template. The PCR reaction was performed for 30 cycles eachconsisting of reaction at 98° C. for 10 sec, 55° C. for 30 sec, and 72°C. for 2 minutes, with Pyrobest DNA polymerase (TaKaRa shuzo). Theamplified DNA fragment and the vector pMW219 were digested with EcoRIand SalI and ligated each other to obtain the plasmid pMW218BSB. It wasconfirmed that there is no error introduced by PCR by sequencing of theamplified fragment. The aforementioned ORF is inserted as reversedirection to the lacZ promoter of the vector.

pMW219BSB was introduced into ME8320 strain in the same manner asdescribed in <2> and plated on LB medium containing 25 mg/L ofkanamycin. Formed colonies were picked up by 10 strains and these wereinoculated and cultured in M9 medium, however, the growth was not found.

<4> Forced Expression of the ORF Having Homology with serB

The inserted fragment in pMW219BSB was changed orientation to be placedforward direction in order to be expressed forcedly. The obtainedplasmid was introduced into ME8320 strain and plated on LB mediumcontaining 25 mg/L of kanamycin. The formed colonis were found to growon the minimal medium.

According to the aforementioned result, it was demonstrated that theaforementioned ORF having homology with serB gene has an ability tocomplement serB deficiency of Escherichia coli. Therefore, it wasconfirmed that the ORF is serB homologue of Brevibacterium flavum.

Another ORF was found just upstream of the ORF having homology with serBgene in the cloned fragment obtained as described in aforementioned <2>.It was thought that pMW219BSB could not complement serB deficiencybecause these ORFs were forming operon and there was no promoter regionand the like just upstream of the ORF having homology with serB gene.

1. An isolated protein which consists of the amino acid sequence of SEQID NO:
 2. 2. An isolated protein which is encoded by a DNA whichconsists of nucleotides 210-1547 of the nucleotide sequence of SEQ IDNO:1 and the protein has phosphoserine phosphatase activity.